RESUMO
Utilizing RNA sequence (RNA-Seq) splice junction data from a cohort of 1841 B-cell acute lymphoblastic leukemia (B-ALL) patients we define transcriptionally distinct isoforms of ARID5B, a risk-associated gene identified in genome wide association studies (GWAS), which associate with disease survival. Short (S) and long (L) ARID5B transcripts, which differ in an encoded BAH-like chromatin interaction domain, show remarkable correlation to the isoform splicing pattern. Testing of the ARID5B proximal promoter of the S & L isoforms indicated that both are functionally independent in luciferase reporter assays. Increased short isoform expression is associated with decreased event-free and overall survival. The abundance of short and long transcripts strongly correlates to B-ALL prognostic stratification, where B-ALL subtypes with poor outcomes express a higher proportion of the S-isoform. These data demonstrate that the analysis of independent promoters and alternative splicing events are essential for improved risk stratification and a more complete understanding of disease pathology.
Assuntos
Processamento Alternativo , Estudo de Associação Genômica Ampla , Humanos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Splicing de RNA , Sequência de Bases , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
IL13Rα2 is an attractive target due to its overexpression in a variety of cancers and rare expression in healthy tissue, motivating expansion of interleukin 13 (IL13)-based chimeric antigen receptor (CAR) T cell therapy from glioblastoma into systemic malignancies. IL13Rα1, the other binding partner of IL13, is ubiquitously expressed in healthy tissue, raising concerns about the therapeutic window of systemic administration. IL13 mutants with diminished binding affinity to IL13Rα1 were previously generated by structure-guided protein engineering. In this study, two such variants, termed C4 and D7, are characterized for their ability to mediate IL13Rα2-specific response as binding domains for CAR T cells. Despite IL13Rα1 and IL13Rα2 sharing similar binding interfaces on IL13, mutations to IL13 that decrease binding affinity for IL13Rα1 did not drastically change binding affinity for IL13Rα2. Micromolar affinity to IL13Rα1 was sufficient to pacify IL13-mutein CAR T cells in the presence of IL13Rα1-overexpressing cells in vitro. Interestingly, effector activity of D7 CAR T cells, but not C4 CAR T cells, was demonstrated when cocultured with IL13Rα1/IL4Rα-coexpressing cancer cells. While low-affinity interactions with IL13Rα1 did not result in observable toxicities in mice, in vivo biodistribution studies demonstrated that C4 and D7 CAR T cells were better able to traffic away from IL13Rα1+ lung tissue than were wild-type (WT) CAR T cells. These results demonstrate the utility of structure-guided engineering of ligand-based binding domains with appropriate selectivity while validating IL13-mutein CARs with improved selectivity for application to systemic IL13Rα2-expressing malignancies.
Assuntos
Imunoterapia Adotiva , Subunidade alfa2 de Receptor de Interleucina-13 , Interleucina-13 , Neoplasias , Animais , Linhagem Celular Tumoral , Humanos , Imunoterapia Adotiva/métodos , Interleucina-13/genética , Interleucina-13/farmacocinética , Interleucina-13/uso terapêutico , Subunidade alfa2 de Receptor de Interleucina-13/antagonistas & inibidores , Camundongos , Neoplasias/terapia , Engenharia de Proteínas , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Malignant brain tumors, including glioblastoma, represent some of the most difficult to treat of solid tumors. Nevertheless, recent progress in immunotherapy, across a broad range of tumor types, provides hope that immunological approaches will have the potential to improve outcomes for patients with brain tumors. Chimeric antigen receptors (CAR) T cells, a promising immunotherapeutic modality, utilizes the tumor targeting specificity of any antibody or receptor ligand to redirect the cytolytic potency of T cells. The remarkable clinical response rates of CD19-targeted CAR T cells and early clinical experiences in glioblastoma demonstrating safety and evidence for disease modifying activity support the potential of further advancements ultimately providing clinical benefit for patients. The brain, however, is an immune specialized organ presenting unique and specific challenges to immune-based therapies. Remaining barriers to be overcome for achieving effective CAR T cell therapy in the central nervous system (CNS) include tumor antigenic heterogeneity, an immune-suppressive microenvironment, unique properties of the CNS that limit T cell entry, and risks of immune-based toxicities in this highly sensitive organ. This review will summarize preclinical and clinical data for CAR T cell immunotherapy in glioblastoma and other malignant brain tumors, including present obstacles to advancement.
Assuntos
Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/terapia , Imunoterapia Adotiva , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Antígenos de Neoplasias/imunologia , Neoplasias Encefálicas/patologia , Engenharia Genética , Humanos , Imunidade , Imunoterapia Adotiva/métodos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos Quiméricos/genética , Resultado do TratamentoRESUMO
Pharmacophore multiplets are useful tools for 3D database searching, with the queries used ordinarily being derived from ensembles of random conformations of active ligands. It seems reasonable to expect that their usefulness can be augmented by instead using queries derived from single ligand conformations obtained from aligned ligands. Comparisons of pharmacophore multiplet searching using random conformations with multiplet searching using single conformations derived from GALAHAD (a genetic algorithm with linear assignment for hypermolecular alignment of datasets) models do indeed show that, while query hypotheses based on random conformations are quite effective, hypotheses based on aligned conformations do a better job of discriminating between active and inactive compounds. In particular, the hypothesis created from a neuraminidase inhibitor model was more similar to half of 18 known actives than all but 0.2% of the compounds in a structurally diverse subset of the World Drug Index. Similarly, a model developed from five angiotensin II antagonists yielded hypotheses that placed 65 known antagonists within the top 0.1-1% of decoy databases. The differences in discriminating power ranged from 2 to 20-fold, depending on the protein target and the type of pharmacophore multiplet used.